Three-dimensional architecture of the outer hair cell lateral wall as determined by electron tomography
Triffo, William Jeffrey
Raphael, Robert M.
Doctor of Philosophy
Cochlear outer hair cells (OHCs) are understood to provide the basis for mechanical amplification in the cochlea through electromotility, a phenomenon in which OHCs change length in response to variations in transmembrane voltage. Thus, study of peripheral hearing and cochlear function is linked to analysis of the OHC's operation as a mechanical actuator. A complete understanding of OHC mechanics depends on precise knowledge of intracellular structure, and efforts to model OHC physiology are likewise limited to the fidelity of known intracellular geometry. Previous studies have localized the mechanism of OHC electromotility to the cortex of the OHC, referred to as the lateral wall. The lateral wall can be viewed as a trilaminate composite made up of (1) the plasma membrane (PM), (2) a network of actin and spectrin termed the cortical lattice, and (3) lamellar stacks known as the subsurface cisternae (SSC). 3D study of lateral wall components in intact cells would augment the existing model, which relies in part on 2D data obtained from partially extracted cell preparations. Electron tomography utilizes a series of transmission electron microscopy projections from a tilted sample to reconstruct a volume density map, imaging macromolecular assemblies in their native cellular context. We began by evaluating TEM sample preparation methods, beginning with conventional aldehyde fixation protocols and progressing to high-pressure freezing and freeze-substitution (HPF/FS), which required development of new methodology for application to OHCs. Using HPF/FS cochlear samples from guinea pig, we employed electron tomography to study the 3D structural relationship of the PM, cortical lattice, and SSC. We observed and characterized novel structure within the SSC membrane and lumen, and also observed physical connections between the circumferential actin filaments and the SSC. Combined with the pillar proteins that join the PM and cortical lattice, these actin-SSC connections provide mechanical coupling between the PM and underlying SSC, which has direct implications for current models of OHC motility.